Journal of Membrane Biology

, Volume 217, Issue 1–3, pp 63–69 | Cite as

Role of the Cytoplasmic Loop Domain of Cx43 in Its Intracellular Localization and Function: Possible Interaction with Cadherin



We have previously shown that intracellular trafficking and function of connexin (Cx) 26 and Cx43 are controlled by E-cadherin. In the present study, we attempted to determine which part of Cx43 is involved in this control mechanism. Since Cx26 has a very short C terminus in the cytoplasm, we hypothesized that the C-terminal domain may not be important for this process and, indeed, found that green fluorescence protein (GFP)-tagged Cx43ΔC (deleted from the codon 239) moved to the plasma membrane both in P3/22(E), a mouse papilloma cell line which expresses E-cadherin, and HeLa cells only at high calcium culture conditions. We then found that the GFP-tagged Cx43(CL 26)ΔC mutant, in which the cytoplasmic loop domain of Cx43 was exchanged with that of Cx26, remains in the cytoplasm in HeLa, HeLaCx43 and P3/22(E) cells, suggesting the importance of the cytoplasmic loop domain. In order to determine which part of the cytoplasmic domain plays a key role, we introduced four deletion mutations (deletion of codons 101–111 [mutant D1], 120–130 [D2], 131–137 [D3] or 146–159 [D4]) to the GFP-tagged Cx43ΔC gene. When these mutants were transfected into HeLa cells, D1 and D4 mutants were localized in the cytoplasm, while D2 and D3 were found in the plasma membrane only in high Ca2+ medium. However, none of these four mutants recovered gap junctional intercellular communication (GJIC). On the other hand, when these mutants were transfected into HeLaCx43 and P3/22(E) cells (which express functional Cx43), D1, D2 and D3, but not D4, moved to the plasma membrane and colocalized with endogenous Cx43 in high Ca2+ medium; all of these mutants showed a dominant negative effect on GJIC in HeLaCx43 cells. Further deletion studies indicated that the critical amino acids involved in this intracellular trafficking of Cx43 lie between codons 100 and 102.


Gap junction Cx43 Connexin Cadherin Protein trafficking 


  1. Bruzzone R, Gomes D, Denoyelle E, Duval N, Perea J, Veronesi V, Weil D, Petit C, Gabellec MM, D’Andrea P, White TW (2001) Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness. Cell Commun Adhes 8:425–431PubMedCrossRefGoogle Scholar
  2. Bruzzone R, White TW, Scherer SS, Fischbeck KH, Paul DL (1994) Null mutations of connexin32 in patients with X-linked Charcot-Marie-Tooth disease. Neuron 13:1253–1260PubMedCrossRefGoogle Scholar
  3. Enomoto T, Martel N, Kanno Y, Yamasaki H (1984) Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP. J Cell Physiol 121:323–333PubMedCrossRefGoogle Scholar
  4. Fu CT, Bechberger JF, Ozog MA, Perbal B, Naus CC (2004) CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression. J Biol Chem 279:36943–36950PubMedCrossRefGoogle Scholar
  5. Giepmans BN, Moolenaar WH (1998) The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr Biol 8:931–934PubMedCrossRefGoogle Scholar
  6. Hernandez-Blazquez FJ, Joazeiro PP, Omori Y, Yamasaki H (2001) Control of intracellular movement of connexins by E-cadherin in murine skin papilloma cells. Exp Cell Res 270:235–247PubMedCrossRefGoogle Scholar
  7. Ise K, Omori Y, Katoh F, Yamasaki H (2004) Control of connexin26 (Cx26)-mediated growth suppression of HeLa cells by the Cx26-specific binding protein, AP26. Proc Am Assoc Cancer Res 45:86Google Scholar
  8. Jongen WMF, Fitzferald DJ, Asamoto M, Piccoli C, Slaga TJ, Gros D, Takeichi M, Yamasaki H (1991) Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin. J Cell Biol 114:545–555PubMedCrossRefGoogle Scholar
  9. Kawasaki Y, Miyoshi S, Yamasaki H (2004) Mechanism of connexin 43-mediated growth control; possible involvement of connexin43-associated proteins. Proc Am Assoc Cancer Res 45:82Google Scholar
  10. Kelsell DP, Dunlop J, Stevens HP, Lench NJ, Liang JN, Parry G, Mueller RF, Leigh IM (1997) Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387:80–83PubMedCrossRefGoogle Scholar
  11. Kostin S, Hein S, Bauer EP, Schaper J (1999) Spatiotemporal development and distribution of intercellular junctions in adult rat cardiomyocytes in culture. Circ Res 85:154–167PubMedGoogle Scholar
  12. Kumar NM (1999) Molecular biology of the interactions between connexins. Novartis Found Symp 219:6–16PubMedCrossRefGoogle Scholar
  13. Laird DW (2005) Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim Biophys Acta 1711:172–182PubMedCrossRefGoogle Scholar
  14. Maass K, Ghanem A, Kim JS, Saathoff M, Urschel S, Kirfel G, Grummer R, Kretz M, Lewalter T, Tiemann K, Winterhager E, Herzog V, Willecke K (2004) Defective epidermal barrier in neonatal mice lacking the C-terminal region of connexin43. Mol Biol Cell 15:4597–4608PubMedCrossRefGoogle Scholar
  15. Matsuda T, Fujio Y, Nariai T, Ito T, Yamane M, Takatani T, Takahashi K, Azuma J (2006) N-cadherin signals through Rac1 determine the localization of connexin 43 in cardiac myocytes. J Mol Cell Cardiol 40:495–502PubMedCrossRefGoogle Scholar
  16. Mege RM, Matsuzaki F, Gallin WJ, Goldberg JI, Cunningham BA, Edelman GM (1988) Construction of epithelioid sheets by transfection of mouse sarcoma cells with cDNAs for chicken cell adhesion molecules. Proc Natl Acad Sci USA 85:7274–7278PubMedCrossRefGoogle Scholar
  17. Mese G, Londin E, Mui R, Brink PR, White TW (2004) Altered gating properties of functional Cx26 mutants associated with recessive non-syndromic hearing loss. Hum Genet 115:191–199PubMedCrossRefGoogle Scholar
  18. Meyer RA, Laird DW, Revel JP, Johnson RG (1992) Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies. J Cell Biol 119:179–189PubMedCrossRefGoogle Scholar
  19. Milks LC, Kumar NM, Houghten R, Unwin N, Gilula NB (1988) Topology of the 32-kd liver gap junction protein determined by site-directed antibody localizations. EMBO J 7:2967–2975PubMedGoogle Scholar
  20. Moreno AP, Chanson M, Elenes S, Anumonwo J, Scerri I, Gu H, Taffet SM, Delmer M (2002) Role of the carboxyl terminal of connexin43 in transjunctional fast voltage gating. Circ Res 90:450–457PubMedCrossRefGoogle Scholar
  21. Moriyama M, Omori Y, Ishizaki Y, Nishikawa Y, Tokairin T, Ogawa J, Enomoto K (2003) Connexin26-mediated gap junctional communication reverses the malignant phenotype of MCF-7 breast cancer cells. Cancer Sci 94:501–507CrossRefGoogle Scholar
  22. Nelis E, Haites N, Van Broeckhoven C (1999) Mutations in the peripheral myelin genes and associated genes in inherited peripheral neuropathies. Hum Mutat 13:11–28PubMedCrossRefGoogle Scholar
  23. Omori Y, Yamasaki H (1998) Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner. Int J Cancer 78:446–453PubMedCrossRefGoogle Scholar
  24. Paznekas WA, Boyadjiev SA, Shapiro RE, Daniels O, Wollnik B, Keegan CE, Innis JW, Dinulos MB, Christian C, Hannibal MC, Jabs EW (2003) Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet 72:408–418PubMedCrossRefGoogle Scholar
  25. Ressot C, Gomes D, Dautigny A, Pham-Dinh D, Bruzzone R (1998) Connexin32 mutations associated with X-linked Charcot-Marie-Tooth disease show two distinct behaviors: loss of function and altered gating properties. J Neurosci 18:4063–4075PubMedGoogle Scholar
  26. Sohl G, Willecke K (2003) An update on connexin genes and their nomenclature in mouse and man. Cell Commun Adhes 10:173–180PubMedCrossRefGoogle Scholar
  27. Toyofuku T, Yabuki M, Otsu K, Kuzuya T, Hori M, Tada M (1998) Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes. J Biol Chem 273:12725–12731PubMedCrossRefGoogle Scholar
  28. Wang HL, Chang WT, Yeh TH, Wu T, Chen MS, Wu CY (2004) Functional analysis of connexin-32 mutants associated with X-linked dominant Charcot-Marie-Tooth disease. Neurobiol Dis 15:361–370PubMedCrossRefGoogle Scholar
  29. White TW (2000) Functional analysis of human Cx26 mutations associated with deafness. Brain Res Brain Res Rev 32:181–183PubMedCrossRefGoogle Scholar
  30. Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, Deutsch U, Sohl G (2002) Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem 383:725–737PubMedCrossRefGoogle Scholar
  31. Yancey SB, John SA, Lal R, Austin BJ, Revel JP (1989) The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. J Cell Biol 108:2241–2254PubMedCrossRefGoogle Scholar
  32. Yeager M, Gilula NB (1992) Membrane topology and quaternary structure of cardiac gap junction ion channels. J Mol Biol 223:92–48CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Chika Nambara
    • 1
  • Yumi Kawasaki
    • 1
  • Hiroshi Yamasaki
    • 1
  1. 1.Department of Bioscience, School of Science and TechnologyKwansei Gakuin UniversitySandaJapan

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